Project summary. Previously we showed that in experimental animal models, the presence of the dsRNA virus LRV1 infecting strains of Leishmania guyanensis confers elevated pathology and metastasis, mediated by a TLR3-dependent inflammatory response. The relevancy of this finding to humans was established by recent findings that patients infected with Leishmania bearing LRV1 show an elevated frequency of drug treatment failures, as well increased pathology and cytokine responses. Here we extend these studies to L. braziliensis, first by developing new animal models and tools. The newly discovered bunyavirus- like virus ?TOP? will be a high priority; TOP occurs in >95% of all L. braziliensis strains examined and the majority of related Viannia species. Extensive preliminary data suggest a strong role in virulence, potentially exceeding that of LRV1. In Aim 1 we will study mechanisms of LRV1- and TOP-dependent virulence. A well-chosen series of isogenic lines showing different combinations of LRV1 and/or TOP (virotypes) will be established and their virulence characterized. These will then be used to probe the mechanism of LRV1-dependent virulence, which is associated with a strong macrophage response similar to that induced by type I interferons. TOP-dependent virulence seems to act through a completely different mechanism, independent of interferon and likely involving dendritic cells. In Aim 2 we focus on molecular virology, especially of TOP, as these studies will likely inform efforts to inhibit these viruses directly. We established that TOP is a highly divergent lineage within a new family of the Bunyavirales termed ?Leishbunyaviridae?. Since these novel features arose precisely at the time deep in evolution when Leishmania transitioned from monxenous to dixenous/vertebrate parasitism, these structural differences are likely to contribute to the TOP pathogenic mechanism. We will explore their coding potential and the existence and role(s) of predicted proteins, and use genetic approaches to functional test their role in viral replication and virulence.